Antibiotic derivatives

ABSTRACT

Derivatives of kanamycin A and B have been prepared which possess substantially improved antibacterial activity. An example of such an agent is 1-(L-(-)- Beta -amino- Alpha hydroxypropionyl)-kanamycin B (IVa, BB-K122).

United States Patent [1 1 Naito et al.

[4 1 Sept. 9, 1975 ANTIBIOTIC DERIVATIVES [75] Inventors: TakayukiNaito; Susumu Nakagawa;

Yoshio Abe, all of Tokyo, Japan [73] Assignee: Bristol-Myers Company,New York,

221 Filed: Feb. 23, 1973 21 Appl. N0.: 335,211

[52] US. Cl 260/210 K; 424/180 [51] Int. Cl. C07G 11/00 [58] Field ofSearch 260/210 AB, 210 K, 210 R [56] References Cited UNITED STATESPATENTS 3,032,547 5/1962 Rothrock et al. 260/210 K 3,541,078 ll/1970 Wooet a1. 260/210 R 3,753,973 8/1973 Umezawa 260/210 K 3,781,268 12/1973Kawaguchi ct al. 260/210 AB Primary ExamiherJohnnie R. Brown AssistantExaminer-Cary B. Owens Attorney, Agent, or Firm-Robert E. Havranek [5 7ABSTRACT 7 Claims, No Drawings ANTIBIOTIC DERIVATIVES BACKGROUND OF THEINVENTION 1. Field of the Invention:

This invention relates to a semisynthetic lsubstituted derivatives ofkanamycin A or B. said cont pounds being prepared by aeylating thel-aminofunction of kanamycin A or B with a B-amino-a-hydroxypropionylmoiety.

2. Description of the Prior' Art:

The kanamycins are known antibiotics described in Merck Index. 8thEdition. pp. 597-598. Kanamycin A is a compound having the formulaKan-amycin B is a compound having the formula The compound designated l[L-( l-y-amino-a-hydroxybutyrylI-kanamycin A {BB-KXlis described in theJournal of Antibiotics. 12). pp. 695-73l (Decemher. W72).

SUMMARY OF THE INVENTION The compound having the formula in which R isOH or NH and R is L-(-)-B-amino-ahydroxypropionyl; or a nontoxicpharmaceutically aceeptable acid addition salt thereofis a valuableantibacterial agent.

This invention relates to semi-synthetic derivatives of kanamycin A andB, said compounds being known as l-[ L-( -)-B-amino-oz-hydroxypropionyll-kan'amycin A and B having the formula hydroxypropionyl; or a nontoxicpharmaceuticalLv acceptable acid addition salt thereof For the purposeof this disclosure. the term nontoxic. pharmaccutically acceptable acidaddition salt shall mean a mono. di-. tri-. tetra or pentasalt formed bythe interaction of one molecule of compound IV with 1-5 moles of anontoxic. pharmaceutically acceptable acid. Included among these acidsare acetic. hydrochloric sulfuric. maleic. phosphoric. nitric.hydrobromic. ascorbic malic and citric acid. and those other acidscommonly used to make salts of amine containing pharmaceuticals.

The compounds ofthe present invention are prepared by the followingdiagraniatic scheme:

2.) Compound ll N-Hydruxysuccinimidu cslur uf l.-()-/3-hunyyloxycurhnnyl- :lminu-B-hydmxypmpiunic acid HO I A preferredembodiment of the present invention is the compound having the formulaHO -NH R in which R is H or R is H or L-()-,8-amino-a-hydroxypropionyland R is OH or NH wherein R or R must be other than H;

or a nontoxic pharmaceutically acceptable acid addition salt thereof.

Another preferred'embodiment is the compound of formula V in which R ismaceutically acceptable acid addition salt thereof.

Another more preferred embodiment is the compound of formula V wherein Ris H, R is L-()-B- amino-ahydroxypropionyl and R is NH. or a nontoxicpharmaceutieally acceptable acid addition salt thereof.

Other most preferred embodiments are the sulfate, hydrochloride,acetate, maleate, citrate, ascorbate, nitrate or phosphate salts ofcompound V.

Another more preferred embodiment is the monosulfate salt of compound V.

Still another preferred embodiment is the disulfate salt of compound V.

The objectives of the present invention have been achieved, by theprovision according to the present invention of the process for thepreparation of the compound having the formula CH -NH R" is OH or NH. ora nontoxic pharmaeeutically acceptable acid addition salt thereof; whichprocess comprises the consecutive steps of A. acylating kanamycin A orkanamycin B with an acylating agent selected from the compounds havingthe formulas (or a carbodiimide thereof) or (or a carbodiimide thereof),in which R and R are alike or different and each is H, F, Cl, Br, N0 OH,(lowcr.)alkyl or (lower)alkoxy, X is chloro, bromo or iodo, or afunctional equivalent as an acylating agent;

in a ratio of one mole or less of acylating agent per mole of kanamycinA or B in a solvent preferably selected from the group comprised ofdimcthylformamide. dimethylacetamide tetrahydrofuran. dioxane.LZ-dimcthoxyethane, methanol. ethanol, water. acetone, pyridine.N-(lower)alkylpiperidine, or mixtures thereof. but preferablydimethylformamide-water at a temperature below 50C. and preferably below25 C to produce the compound having the formula in which Y is a radicalof the formula 0 CH O in which R. R and R are as defined above;

B. acylating compound I] with an acylating agent having the formula inwhich W is a radical selected from the group comprising x-wn butpreferably M is a radical selected from the group comprising O-i l N butpreferably --ON III in which R. Y and W are as above: and

C. removing the blocking groups W andY from compound III by methodscommonly known in the art. and

preferably when W and Y are radicals of the formula by hydrogenatingcompound lll with hydrogen in the presence of a metal catalyst.preferably selected from the group comprising palladium. platinum. Raneynickel. rhodium. ruthenium and nickel. but preferably palladium. andmost preferably palladium on charcoal. in a water-water miscible solventsystem. preferably selcctcd from the groupcomprising water and dioxanc.tetrahydrofuran. ethyleneglycol dimethyl ether. propyleneglycol dimethylether; or the like. but preferably 121 water tetrahydrofuran to producethe com pound of formula IV.

It should be apparent to those knowledgeable in the art that otheragents can be used in the process above to acylate the amine functionsof the intermediate compounds of the instant invention. This disclosureis meant to include all such acylating agents that produce labile amineblocking groups. said labile blocking groups commonly employed. in thesynthesis of peptides. The labile blocking groups must be readilyremovable by methods commonly known in the art. Examples of said labileblocking groups and their removal can be found in the review of A.Kapoor. J. Pharm. Sciences. 59, pp. 1-27 (1970). Functional equivalentsas acylating agent for primary amine groups would include correspondingcarboxylic chlorides. bromides, acid anhydrides, including mixedanhydrides and particularly the mixed anhydrides prepared from strongeracids such as the lower aliphatic monoesters of carbonic acid. of alkyland aryl sulfonic acids and of more hindered acids such asdiphenylacetic acid. In addition. an acid azide or an active ester ofthioestcr (e.g., with p-nitrophenol, 2.4-dinitrophenol, thiophenol.thioacetic acid) may be used or the free acid itself may be coupled withthe kanamycin derivative (ll) after first reacting said free acid withN.Ndimethylchloroforminium chloride Icf. Great Britain l.()()8.l7() andNovak anc Wcichet. Experientia XXI/6, 360 W65 or by the use of enzymesor of an N.N'-carbonyldiimidazole or ar N N-earhonylditriazole lcf.Sheehan and Hess, Amer. Chem. Soc. 77. I067. (1955)] or of alkynylaminereagent lcl'. R. Buijilc and H. G. Viche. Angew Chem.. InternationalEdition 3. 582 (1964)]. or of: ketenimine reagent lcf. C. L. Stevens andM. E. Monk J. Amer. Chem. Soc. 83. 1010 1961 )1. Another equiv alent ofthe acid chloride is a corresponding azolide i.e.. an amide of thecorresponding acid whose amid nitrogen is a member of a quasiaromaticlive mem bered ring containing at least two nitrogen atoms. i.e.imidazolc. pyrazole. the triazoles. benzimidazolc. ben zotriazole andtheir substituted derivatives. As an ex ample of the general method forthe preparation of an azolide, N.N'carbonyldiimidazolc reacted with acarboxylic acid in equimolar proportions at room temperature intetrahydrofuran. chloroform, dimethylformamide, or a similar inertsolvent to form the carboxylic acid imidazolide in practicallyquantitative yield with liberation of carbon dioxide and one mole ofimidazole. Diearboxylic acids yield diimidazolides. The byproduct,imidazole. precipitates and may be separated and the imidazolidcisolated, but this is not essen tial. These reactions are well-known inthe art (cf. US. Pat. Nos. 3,079.3 l4, 3,l 17.126 and 3.129.224 andBritish Pat. Nos. 932,644. 957,570 and 959,054).

TABLE I (MIC lug/ml.) BB-K l] Kanamyein MIC (meg/ml.) (lVa) A If. (u/iNlHJ 1.6 0.8 Juhl Alll9 1. 6 l.6 Al5l59 1.6 [.6 lx'M-R A2036} 1.6 l00A9844 0.8 0.8

" KM-R A20365 0.4 100 K-IZ L6 0.8 KM-R A20664 L6 6.3

" KM-R A20665 0.8 I00 W677 I v A20684 L6 0.8 JR/W677 A2068} l.6 I00 I\pm'umnnim- D-l l 5 i 0.4 0.2 'l'ypc 22 No. 3038 A20680 1.6 -l00 .s'.nmrt'lzvu'ln A2001) 1.6 I6 I. m'rngi/mn/ D- [5 3.l ll5 H9 l)-l l3 KM-R6.3 7 l0() Al5l5ll 6.3 H10 A l 5 I94 3.l (EM-R A207l7 6.3 (BM-R A207l86.3 50 I. I'll/guru A9436 0.4 0.4 A9526 0.4 0.4 I. HlirnI i/h A9554 0.80.8

" A9900 0.8 0.8 I. [um-gum! A9553 0.8 0.8 A2003l 0.8 0.8 S. (Ill!('ll.\Mull/r 0.4 0.4 209P SM-R l.6 l.6 KM-R A20239 I6 I00 .lhwlnu'n'rinm 6070.8 0.4 KM-R I00 KhLSM-R I00 T l00 [l/I/(l 0.4 0.4 mum 0.8 0.4

KM-R is lomznntuin resistant. (iM'R l gcnlnlnicin resistant SM-R is\II'LPIUHQCII! resistant.

TABLE [I (Ml(s rug/ml. BB-K I 22 Kanamvcin Ml(' (meg/ml.) (l\'b) BB-Kl23BB-Kl24 I'.'. (uli NlHJ 08 4 l 3.] 0,4 .luhl

Al5l l9 0.8 6.3 3.] 0.8 Al5l69 08 ll 3.1 0.8 KM-R Alum} (is 25 3.! I00A9844 0.8 3 l 3.l 0.4 KMR A20365 0.2 6.3 [.6 50 K ll 0.4 3.l 3.1 0.4KM-R A20664 0.4 6.3 3 l 0.8 KM-R A2066 0.: 6 3 L6 25 W677 Alums-t 0,4 33 s l 3 TABLE II Cntinued Eli-K122 Kanamycin Ml( (meg/ml.) (lVh)BEE-K123 BER-K124 B JR/W677 A20683 1.6 50 3.1 100 K. pm'umnniut' D-l l 02 1.6 0.8 0.1

Type 22 3038 A20680 1.6 50 6.3 100 S. Hmrcurw'nx A2001) 1.6 6.3 3.1 0.8P. acru g'inuxu D- 0.8 25 3.1 6.3 D-l 13 KM-R 12.5 100 2100 A9923 3.1100 12.5 25 A9930 0.4 12.5 1.6 6.3 P. m'ruginm'u A15150 1 3.1 100 25 25A15195 1.6 100 6.3 12.5 GM-R A20717 3.1 100 25 50 "GM-R P. rulgurixA9436 0.2 1.6 1.6 0.2 A9526 0.4 3.1 3.1 0.2 P. "rim/ ill's A9554 0.8 3.16.3 0.4 A9900 0.8 3.1 3.1 0.8 I. murgunl'l' A9553 0.8 6.3 6.3 0.8 A2003]0.8 6.3 6.3 0.8 S. uurwm' Smith 0.2 0.8 0.8 0.1 2091 SM-R 0.8 3.1 6.30.8 "KM-R A2023) 0.8 25 6.3 50 My'uliut'lcrium r 607 0.8 6.3 3.1 1.6KM-R 100 100 100 100 KM.$M-R 100 100 100 100 p/llr'i 0.8 6.3 1.6 1.6I'tllltlt' 0.8 6.3 3.1 1.6

The above MIC data show that compounds IVa and IVb are substantiallymore active against a number the test organisms. particularly thepseudomonas and other kanamycin resistant organisms.

The compounds IV are valuable as antibacterial agents. nutritionalsupplements in animal feeds. therapeutic agents in poultry and animals,including man.

and are especially valuable in the treatment of infcc- 4 tious diseasescaused by Gram-positive and Gramnegative bacteria.

The compounds 1V when administered orally are useful as an adjunctivetreatment for preoperative steril- 4S ization of the bowel. Both aerobicand anaerobic flora which are susceptible to these drugs are reduced inthe large intestine. When accompanied by adequate mechanical cleansing,they are useful in preparing for colonic surgery.

EXAMPLES EXAMPLE 1 Preparation of L-B-Benzyloxycarbonylaminou hdroxypropionic Acid (V1).

L-B-Amino-a-hydroxypropionic acid* (8.2 g. 0.078 mole) was dissolved ina solution of 6.56 g. (0.0164 mole) of sodium hydroxide and in 60 ml. ofwater. To the stirred solution was added dropwise 14.7 g. (0.086 mole)of carbobenzoxy chloride below 5 C. The mix ture was stirred for an hourat room temperature. washed with 60 ml. of ether and adjusted to pH 2with dilute HCl. The precipitate was collected by filtration. washedwith water and air-dried to give 9.65 g. (52 percent) of V1. Thefiltrate was extracted with five -ml. portions of ether. The etherealsolution was washed with water. dried over sodium sulfate and evaporatedto dryness in vacuo to give additional 2.0 g. l l percent) of VI. Atotal of l 1.65 g. of VI was crystallized from 500 ml. of benzene-ethylacetate (4:1) to give 9.36 g. (50 percent) of pure V1. m.p. 128.5-129.5C. Infrared (IR) (KBr): 3 1745. 1690 cm". [01],? +2.9 (e 5.0. MeOH).Nuclear Magnetic Resonance Spectra [NMR (DMSO-d 1: 7 (in ppm) 3.05-3.45(2H. m, CH- N), 4.05

EXAMPLE 2 N-Hydroxysuccinimide Ester ofL-B-benzyloxycarbonylamino-a-hydroxypropionic Acid V11).

To a chilled and stirred solution of 478 mg. (2 m.moles) of V1 and 230mg. (2 m.moles) of N- hydroxysuccinimide in 10 ml. of tetrahydrofuran(THF) was added 412 mg. (2 m.moles) of dicyclohexylcarbodiimide. Themixture was stirred for an hour at 5 C.. for 2 hours at room temperatureand then filtered to remove the N.N'-dic elohexylurea. The filtratecontaining VI] was used for the next reaction without isolation.

EXAMPLE 3 Preparation of l-[ L-(-)-B-amino-a-hydroxypropionyll-kanamycinA (lVa. BB-Kl0l To a stirred solution of 1.48 g. (2.4 m.moles) of6-carbobenzoxykanamycin A (lla) in 25 ml. of water- THF (4:1 was addeddropwise the solution of VI] at 5 C. The mixture was stirred for twohours at room temperature and filtered to remove a small amount ofinsoluble material. The filtrate was hydrogenated overnight with 300 mg.of percent palladium on charcoal at room temperature at atmosphericpressure and then filtered to remove the catalyst. The filtrate wasconcentrated in vacuo to remove most of the organic solvent. Theresultant aqueous solution was adjusted to pH 7 with dilute HCl andpassed through a column of Amberlite CG-50 (NH, .80 ml.)**. which waswashed with 200 ml. of water and then eluted with 400 ml. of 0.1 N. 870ml. of0.2 N. and 430 ml. of 0.5 N NH.,OH. The eluate was collected inIO-ml. fractions. Fraction numbers 95l06 which showed activity againstPseudomonas aeruginosa A9843 and Rf values at 0.25 and 0.41 (desiredproduct) by thin layer chromatography (TLC) on a silica gel plate (S-110. ninhydrin)*** were pooled. evaporated. in vacuo and freeze-dried togive 170 mg. of crude lVa.

The crude lVa( 162 mg.) was adsorbed on a column of (C-50(cupro-ammonium form*. ml.). which was washed with 50 ml. of water andeluted with l L of 0.2 N. 700 ml. of 0.5 N and finally 400 ml. of 1.0 NNH.,OH. The eluate was collected in 7-ml. fraction. Tube numbers 269282which showed a single spot at Rf0.4l was combined, evaporated in vacuoand lyophilized to give 79 mg. of copper complex of [Va The copper wasremoved by column chromatography on CG-SO (NH,,', 5 ml.) using 0.2 N NH,OH as an eluent. The eluate was collected in l0-ml. fraction. Tubenumbers 210 were combined. evaporated in vacuo and lyophilized to give26 mg. (2 percent based on VI of lVa. m.p. 200 "205 C. IR (KBr): 1640,1540 cm".

Anal. calcd. for C H N O,;,.2H CO .3H O: C. 36.84; H. 6.84; N. 9.34.Found: C. 36.93; H. 6.00; N, 9.62.

*The cupro-ammonium form of CG-50 was prepared in the following way: toa stirred suspension of CG-50 (NHfi) in water was added 10 percentcupric sulfate solution to give copper salt of CG-50 which was filtered.The resin was washed several times with water. then treated with 1N NHOH under stirring. filtered and washed several times with water to givedeep blue. eupro-ammonium form of CG-50.

**Amberlite CG-50 is the tradename for the chromatographic grade of aweakly acidic cationic exchange resin of a carboxylic-polymethacrylictype.

***Tl..C: silica gel plate. CHCl;,-MeOH-287( NH,OH-H O (1:4:2z1).

EXAMPLE4 Preparation of N-( Benzyloxycarbonyloxy )succinimide.

N-hydroxysuccinimide' (23 g.. 0.2 mole) was dissolved in a solution of 9g. (0.22 mole) of sodium hydroxide in 200 ml. of water. To the stirredsolution was added dropwise 34 g. (0.2 mole) of carbobenzoxy chloridewith water-cooling and then the mixture was stirred at room temperatureovernight to separate the carbobenzoxy derivative which was collected byfiltration. washed with water and air-dried. Yield 41.1 g. (82 percent).Recrystallization from benzene-n-hexane 10:1 gave colorless prismsmelting at 78-79 C. 1. G. W. Anderson et al.. .I. Am. Chem. Soc.. 86.1839 EXAMPLE 5 Preparation of 6-Carbobenzoxykanamycin A Ila).

A solution of 42.5 g m.moles) of kanamyein A free base in 450 ml. ofwater and 500 ml. of dimethylformamide (DMF) was cooled below 0 C. .andstirred vigorously. To the solution was added dropwise over a period ofabout two hours a solution of 22.4 g. (90 m.mole) of N(benzyloxycarbonyloxy )succinimide in 500 ml. of DMF. The mixture wasstirred at l0 to 0 C. overnight and then at room temperature for oneday. The reaction mixture was evaporated under reduced pressure belowabout 50 C. The oily residue was dissolved in a mixture of 500 ml. waterand 500 ml. butanol. the mixture being filtered to remove insolublematerial and separated into two layers. The butanol and aqueous layerswere treated with butanol-saturated water (500 ml. X 2) andwater-saturated butanol (500 ml. X 2). respectively. using a techniquesimilar to counter current distribution. The three aqueous layers werecombined and evaporated to dryness under re' duced pressure to give anoily residue. at part of which crystallized on standing at roomtemperature. To the residue including the crystals was added about ml.of methanol. which dissolved the oil and separated it from the crystals.After adding about 300 ml. of ethanol. the mixture was kept at roomtemperature overnight to give a crystalline mass which was collected byfiltration. lt weighed 44 g. The product. contained a small amount ofkanamycin A as indicated by thin layer chromatography usingn-propanolpyridine acetic acidwater 15: 10:3: 1 2) as the solvent systemand ninhydrin the spray reagent.

The crude product was dissolved in 300 ml. of water and chromatographedon a column (30 mm. diameter) of CG-5O ion-exchange resin (NH. 1 15type. 500 ml.). The column was irrigated with 0.1 N ammonium hydroxidesolution and the eluate was collected in TLC (silica gel Rf value F251:ninhydrin) o-Chz-Kana- Kana Solvent System mycin A mycin An-ProHPyridine-AcOH-H O 0.42 0.33 0.4 0.04

(15:10:3112) (main) minor Acetone-AeOHH O 0.24 0.14

AcOMc'n-PrOH-(1NH OH 022* 0.04*

*Deteeted by anlhrone-sulfuric acid.

The final product was found to be accompanied by two minor components byTLC with one of the solvent systems tested. However. the final productwas used without further purification for the preparation of BB- KlOl1Va).

EXAMPLE 6 Preparation of 6-Carbobenzoxykanamycin B (11b).

To a chilled solution of 8.1 g. (0.0168 mole) of kanamycin B in 120 ml.of water and 80 ml. of 1,2- dimethoxyethane was added dropwise withstirring a solution A of 4.2 g. (0.0168 mole) of N-(benzyloxycarbonyloxy)suceinimide in 40 ml. of 1,2 dimethoxyethane. Thereaction mixture was stirred overnight and evaporated under reducedpressure. The residue was dissolved in 100 ml. of water and shaken twicewith 50 ml. of water-saturated n-butanol. The aqueous layer wasseparated and adsorbed on a column of 100 ml. of CG-SO (NHftype). Thecolumn was washed with 200 ml. of water. eluted with 0.05 N NH OH. Theeluate was collected in -ml. fraction. Fractions 121 to 180 werecollected. evaporated and freeze-dried to give 1.58 g. percent) of thedesired product. Fractions 1 to 120 were evaporated andrechromatographed on (G-50 (NHf) to give 1.21 g. 12 percent) of theproduct (11b). M.p. l5l152 C. (dec) |a],, +l04 (C. 2.5. H 0). 3 -=,,l7l0emf.

Anal. called. for C H N O C. 50.56; H, 7.()2: N. 11.34. Found: C, 50.71;H, 7.38:1. 11.48.

TLC (silica gel F254). RF 0.03 in nPrOH-pyridine- AcOH-H O 15:10:3212);Rf 0.16 in acetone-AcOH- H O (:6:74). 1

EXAMPLE 7 Preparation of l-[ L-()-B-Amino-oz-hydroxypropionyl]-kanamycin B [BB-K122(1Vb)].

To a stirred solution of 1.23 g. (2.0 m.moles) of 11b in 20 ml. of waterwas added dropwise a solution of V11 prepared from 478 mg. (20 mmoles)of V1 in 20 ml. of THF at room temperature. The mixture was stirredovernight and then hydrogenated overnight over 300 mg. of 10 percentpalladium on charcoal at room temperature at atmospheric pressure. Thehydrogenated mixture was filtered and the filtrate was concentrated invacuo to remove most of the organic solvent. The resultant aqueoussolution was adjusted to pH 7 with l N hydrochloric acid and adsorbed ona column of CG-50 (NHJ, 40 ml.) which was washed with ml. of water andthen eluted with 900 ml. of 0.1 N and 1.2 L of 0.2 N NH OH. The eluatewas collected in 10-ml. fraction. monitored by ninhydrin spot test. TLCwith S-1 10 system and disk assay using Psemlommlus ucruginusu A9843.and cut into the following appropriate fractions. Each fraction wasevaporated in vacuo and freezedried.

Frac- 'l'uhe tion No. NH ()H( N Weight ldentit 1 91-1 13 0.2 420 mg.kanamycin B 2 121-143 353 mg. Eli-K122 (1\h) BB-Kl23 k knnamycin B 3157-196 ho mg. BB-K l 24 (d1- aeylated compound) Frac- Tube tion No. NH()H( N) Weight Identity 1 191-240 1.0 126 mg. copper complex of [HZ-K1232' 331-360 1.5 mg. copper comple\ of the desired Ell-K122 3' 361 385 41mg. copper complu o1 kananucin B Chromatography of fraction 2' (80 mg.)in order to remove copper afforded 35 mg. (3 percent) of BB K122 (lVb).

1n the same way 27 mg. (2.4 percent) of BB-Kl23 was obtained from 126mg. of fraction 1'.

The physico-chemical data of these compounds are given in the followingTable.

BB-K RlWS-l ll). No. IR (cm (Mp. 1C.) ninhydrin) l2. l64() l57()IQZ-lJfi 0.41 123 l64(l,l57() l77-l80 (1.35

0.21 (main). i24 1650,1550 um MICROANALYSIS DATA and BB-Kl24 is adiacylated derivative of kanamycin B. The compounds have weakantibacterial activity compared to kanamycin B and BB-Kl22 (lVb). SeeTable II 7' 7 EXAMPLE 8 Preparation of the Monosuifate Salt of l-[L-(-)-fi amino-a-hydroxypropionyl l-kanamycin A or B.

One mole of l-[ L-()-,8-amino-u-hydroxypropionyll-kanamycin A or B isdissolved in 1 to 3 liters of water. The solution is filtered to removeany undissolved solidsfTo the chilled and stirred solution is added onemole of sulfuric acid dissolved in 500 ml. of water-.The mixture isallowed to stir for 30 minutes, following which cold ethanol is added tothe mixture till precipitation occurs. The solids are collected byfiltration and are determined to be the desired monosulfate salt. 1

EXAMPLE 9 Preparation of the Disulfate Salt of l-[L-(-)-B-amino-a-hydroxypropionyl ]-kanamycin A or B.

35 grams of 1-[ L-()-,8-amino-cx-hydroxypropionyl] kanamycin A or B (asthe monobicarbonate trihydrate) is dissolved in 125 ml. ofdcionizedwater. The pH is adjusted to 7-7.5 with 50 percent V/V sulfuric acid.

Eight and one half grams of Darco G-60 (activated charcoal) is added andthe mixture is slurried for 0.5 hour at ambient room temperature. Thecarbon is removed by suitable filtration and washed with 35 ml. ofdeionized water. The water is added to the filtrate.

The combined filtrate-wash is adjusted to pH ll.3 with 50 percent V/Vsulfuric acid. This solution is added with rapid stirring over aten'minute period to 60()800 ml. of methanol (3-4 volumes of methanol).The mixture is stirred for five minutes'at pH ll.3,

passed through a 100 mesh screen, stirred for two min-.

ably filtered, washed with 200 ml. of methanol and vacuum dried at 50C.for 24 hours to yield the appropriate disulfate salt.

in which R is H, R is L-()B-amino-a-hydroxypropionyl and R is OH or NHor a nontoxic pharmaceutically acceptable salt thereof.

2. The compound having the formula in which R isL-(.)-B-amino-ahydroxypropionyl; or a nontoxic pharmaceuticallyacceptable salt thereof.

3. The compound having the formula CH lilll in which R isL-(-)-/3-amino-a-hydroxypropionyl; or a nontoxic pharmaceuticalLvacceptable salt thereof.

4. The mOllQSUlftltC salt of the compound of claim 2. 5. The monosulfatesalt. of the compound of claim 3. 6. The disulfate salt of the compoundof claim 2. 7. The disulfate salt of the compound of claim 3.

' :1: :s s: k =l

1. A COMPOUND HAVING THE FORMULA
 2. The compound having the formula 3.The compound having the formula
 4. The monosulfate salt of the compoundof claim
 2. 5. The monosulfate salt of the compound of claim
 3. 6. Thedisulfate salt of the compound of claim
 2. 7. The disulfate salt of thecompound of claim 3.